The estrogen receptor is a multidomain, ligand-activated transcription factor; while the structure of the DNA-binding domain is known in detail from NMR and X-ray studies, the structure of the hormone binding domain is so far unknown. The overall goal of this project is to determine the 3-D structure of this domain, using three approaches that operate in a synergistic fashion: (a) the expression of the hormone binding domain of the estrogen receptor (ER-HBD) in chemically pure and conformationally homogeneous form, (b)the use of specific chemical probes and expression of defined fragments to study structure and function, and (c) the application of computational modeling methods. As Specific Aim I, we will use the pTrxFus vector in E. coli to express the ER N304-S554 sequence (that represents the core of the ER-HBD), and we will use electrospray ionization mass spectrometry (ESI-MS) to certify the identity and chemical purity of the expressed material, which has proved to be a problem in many cases. In Specific Aim 2, we will use this material to obtain topological information on the ER-HBD, by affinity labeling to identify ligand contact sites in the HBD and by residue specific modification to identify exposed sites, and we will probe the core structure of the ER-HBD by expressing HBD fragments and subdomain structural elements, following structure formation by circular dichroism (CD) and function by ligand binding. As Specific Aim 3, we will use advanced computational homology modeling approaches to predict and refine a 3-D structural model of the ER-HBD, using energy-based and pattern recognition methods and testing consistency with results from our own studies; we will use this model to evaluate ligand binding specificity for structure-based design and to plan specificity re-engineering experiments by subdomain swapping. As Specific Aim 4, we will prepare isotopically labeled ligands and ER-HBD core structural fragments for NMR studies, to be done in collaboration with A. J. Wand, Dept. Biochemistry. For X-ray analysis to be done with A. Wang, Depts. Cell and Structural Biology, and Chemistry, we will use preparations that are chemically pure and conformationally homogeneous, and contain heavy atoms in the protein or complexed ligand. We anticipate that these studies, which combine synthetic chemical, biochemical, molecular biological, and (through collaborations) advanced computational modeling, NMR and X-ray structural analysis, will lead to significant advances in our understanding of the structure of the estrogen receptor and its interaction with estrogen hormones.